Wound healing

ABSTRACT

The present invention relates to the use of p38 MAP kinase inhibitors and p38 MAP kinase inhibition to promote wound healing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 ofPCT/US2005/030930 filed Aug. 31, 2005, which claims priority from U.S.60/606,552, filed Sep. 1, 2004.

All documents cited herein are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to the use of p38 MAP kinase inhibitorsand p38 MAP kinase inhibition to promote wound healing.

BACKGROUND OF THE INVENTION

In mammals, injury triggers an organised complex cascade of cellular andbiochemical events that result in a healed wound. Wound healing is acomplex dynamic process that results in the restoration of anatomiccontinuity and function; an ideally healed wound is one that hasreturned to normal anatomic structure, function and appearance.

Mark R. Frey, Anastasia Golovin, and D. Brent Polk (2004) J. Biol.Chem., 10.1074/jbc.M406253200 (published online ahead of print Aug. 16,2004) “EGF-stimulated intestinal epithelial cell migration requires Srcfamily kinase-dependent p38 MAPK signaling” discloses that genetic orpharmacological blockade of p38 signaling in the intestinal epitheliuminhibited the ability of EGF to accelerate wound closure.

The purpose of this invention is to actively promote/enhance the woundhealing process, as delayed or compromised wound healing remains asignificant unmet need.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery that p38 MAPkinase inhibitors promote wound healing. Thus, the rate of wound healingor the extent of wound healing may be achieved through the use of p38MAP kinase inhibitors. It is believed that the enhanced wound healingwill be achieved by the inhibition of the p38 MAP kinase pathway.

A first aspect of the invention provides the use of at least one p38 MAPkinase inhibitor in the manufacture of a medicament for promoting woundhealing.

A second aspect of the invention provides a method of promoting woundhealing in a patient the method comprising administering atherapeutically effective amount of at least one p38 MAP kinaseinhibitor.

A third aspect of the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of at least one p38 MAPkinase inhibitor. Preferably, the pharmaceutical composition is fortopical application.

In a preferred embodiment of the invention there is provided a wounddressing comprising a therapeutically effective amount of at least atone p38 MAP kinase inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Effect of p38 MAP kinase inhibitor (20 μg) or vehicle [pH 4.0Citrate Buffer] treatments on the percentage contraction of a human,adult, fibroblast populated collagen lattice. Data are presented as themean of three replicates ± the standard error of the mean.

DETAILED DESCRIPTION

Therapeutic uses of MAP kinase inhibitors have been described innumerous patent and literature publications: EP1205478A1 (Greenberg,Andrew S. MAP Kinase inhibitors in the treatment of disorders caused byTNF-alpha induced lipolysis; EP1071429A1—Ohkawa, Shingenori. P38 MAPKinase inhibitors; US20020103245A1—Goldstein, David Michael et al.Pyrazole derivatives p38 MAP Kinase inhibitors;US20020156114A1—Goldstein, David Michael et al. Pyrazole derivatives p38MAP Kinase inhibitors; US20030018051A1—Goldstein, David Michael et al.Pyrazole derivatives—p38 MAP Kinase inhibitors; US20030139462A1—Cheng,Soan et al. P38 MAP Kinase inhibitors; US20030212138A1—Obukowicz et al.Combinations of peroxisome proliferator-activated receptor-alphaantagonists and cyclooxygenase-2 selective inhibitors and their use;U.S. Pat. No. 6,444,696B1—Goldstein, David Michael et al. Pyrazolederivatives p38 MAP Kinase inhibitors; U.S. Pat. No. 6,479,507B2—Cheng,Soan et al. P38 MAP Kinase inhibitors; U.S. Pat. No. 6,509,361B1—Weieret al. 1,5 Diaryl substituted pyrazoles as P38 MAP kinase inhibitors;U.S. Pat. No. 6,630,485B2—Cheng, Soan et al. P38 MAP Kinase inhibitors;WO0135959A1—Ingelman-Sundberg, Magnus. Use of thiazole derivatives forthe treatment of p38 mediated disorders; WO2003059294A2—Obukowicz et al.Combinations of peroxisome proliferator-activated receptor-alphaantagonists and cyclooxygenase-2 selective inhibitors and their use;U.S. Pat. No. 6,277,989 B1—Chakravarty et al. Quinazoline derivatives asmedicaments; U.S. Pat. No. 6,410,540 B1—Goehring et al. Inhibitors ofp38alpha kinase; U.S. Pat. No. 6,476,031 B1—Chakravarty et al.Quinazoline derivatives as medicaments; U.S. Pat. No. 6,541,477B2—Goehring et al. Inhibitors of p38alpha kinase; U.S. Pat. No.6,696,443 B2—Mavunkel et al. Piperidine/piperazine-type inhibitors ofp38 kinase; WO00/12074—Goehring et al. Inhibitors of p38alpha kinase;WO00/12497—Chakravarty et al. Quinazoline derivatives as medicaments;WO00/71535 A1—Mavunkel et al. Indole-type derivatives as inhibitors ofp38 kinase; WO01/64676 A2—Goehring et al. Inhibitors of p38alpha kinase;WO02/42292—Dugar et al. Inhibitors of p38 kinase; WO02/44168 A2—Dugar etal. Indole-type Inhibitors of p38 kinase; WO 02/46158 A2—Dugar et al.Piperidine/piperazine-type inhibitors of p38 kinase; and U.S. Pat. No.5,965,583—Beers et al. Substituted imidazoles useful in the treatment ofinflammatory disease).

Whilst the above publications document various medicinal uses of p38 MAPkinase inhibitors (osteoporosis, osteoarthritis, allergic inflammation,periodontal disorder, inflammatory bowel disorder, septic shock,insulin-dependent diabetes mellitus, non-insulin-dependent diabetes,cachexia, pulmonary fibrosis, myasthenia gravis, Crohn's disease,hepatitis, primary biliary cirrhosis, acute pancreatitis, allographrejection, glioblastoma, alopecia areta, psoriasis, ischemia, congestiveheart failure, restenosis, atherosclerosis, systemic lupuserythematosus, nephritis, Guillain-Barre Syndrome, viral myocarditis,HIV replication, T-cell depletion in HIV infection, cognitive deficitsinduced by neuronal inflammation, multiple sclerosis, stroke,neuropathic pain, HIV dementia and Alzheimer's disease) the prior artfails to disclose that p38 MAP kinase inhibitors may advantageously beused to promote wound healing.

In fact, inhibition of the p38 MAP kinase pathway by p38 MAP kinaseinhibitors appears to inhibit keratinocyte migration and cellularcontraction, suggesting that this class of compounds may have a negativeimpact on wound healing. For example, Lee et al. (2000) ExperimentalCell Research, 257, 190-197 suggests that inhibition of p38 MAP kinaseor the addition of a p38 MAP kinase inhibitor to an in vitro modelcauses a delay in contraction. In contrast our data, in vitro and in acompromised healing animal model suggest an enhancement of wound healingwith p38 MAP kinase inhibitors.

Accordingly, a first aspect of the invention provides the use of a p38MAP kinase inhibitor in the manufacture of a medicament for promotingwound healing.

A second aspect of the invention provides a method for promoting woundhealing in a patient the method comprising administering atherapeutically effective amount of a p38 MAP kinase inhibitor.

A third aspect of the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a p38 MAP kinaseinhibitor.

In a preferred embodiment of the invention there is provided a wounddressing comprising a therapeutically effective amount of at least atone p38 MAP kinase inhibitor.

A therapeutically effective amount is that amount necessary toreproducibly increase the rate of wound healing or to increase theextent of wound healing.

The p38 MAP Kinase Inhibitor

Four isoforms of p38 MAP kinase have been identified and are designatedas p38 alpha, beta, delta and gamma (Jiang, Y. et al., J. Biol. Chem.271: 17920-17926 (1996); Kumar, S. et al., Biochem. Bioplzys. Res. Comm.235: 533-538 (1997); Stein, B. et al., J. Biol. Chem. 272: 19509-19517(1997); Li, Z. et al., Biochetn. Biophys. Res. Comm. 228: 334-340(1996); Wang, X. et al., J. Biol. Chem. 272: 23668-23674 (1997)). Theseisoforms differ in tissue expression patterns, substrate utilization,response to direct and indirect stimuli, and susceptibility to kinaseinhibitors. p38 activity is normally low or absent in cells and isinduced selectively by cytokines and environmental stresses such asosmotic shock, heat shock or UV light.

Various p38 MAP kinase inhibitors may be used in the various aspects andembodiments of the invention. These include, and are not limited to, thefollowing classes of compounds: fused-ring heterocycles, biphenylcompounds, phenyl compounds, heterocycles and ureas. Examples offused-ring heterocycles include, and are not limited to, aryl ketonepyrrolo-triazines, azaindoles, benzimidazoles, benzothiazoles,benzimidazolones, benzotriazoles, bicyclic oxopyridine andoxopyrimidines, bicyclic pyridine and pyrimidines, dihydroquinazolinones, dihydropyridopyrimidinones,dihydrothieno[2,3-b]pyridines, fused heteroaryl compounds,heteroalkylamino-substituted bicyclic nitrogen heterocycles, imidazofused compounds, imidazopyridinones, indole-type compounds, pyridazinylpyrazoles, pyridinylimidazoles, pyrido-pyrimidines, pyridyl-1,3-azoles,pyrimidinyl pyrazoles, pyrimidinyloxazole and imidazoles, quinazolines,quinoline and isoquinoline n-oxides, substituteddihydropyrimidopyrimidinones, substituted imidazopyrimidines, thiazoleand imidazo[4,5-b]pyridines, triazolopyridines, and substitutedimidazopyridines.

Examples of biphenyl compounds include, but are not limited to,carbamoyl biphenyl carboxamides, heteroaryl substituted biphenylcompounds, oxadiazolyl biphenylcarboxamides, biphenylcarboxylic amidesand oxadiazolyl-biphenylcarboxamides.

Examples of phenyl compounds include, but are not limited to,aminobenzophenones and aminobenzoylpyridines, aminophenyl ketones,benzamides and benzophenones.

Examples of heterocycles include, but are not limited to,imidazolinones, tetrasubstituted imidazoles, substituted isoxazoles,piperidin-2-ones, arylmethoxypyrazines, pyrazoles, pyrazole derivatives,pyridinyl pyrazoles, aminopyrazoles, substituted pyrazoles, substitutedpyridazinones, aroyl pyridinones, arylmethoxypyridines, nicotinamides,substituted pyridinones, disubstituted pyridine, pyrimidine, pyridazineand triazines, pyridine, pyrimidines, aminopyrroles, substitutedpyrroles, thiazoles, substituted triazoles and triazole amides.

Examples of ureas, include but are not limited to, aryl ureas, carboxyaryl substituted diphenyl ureas, heteroaryl ureas and pryrazolylnaphthalene ureas.

In one embodiment, a p38 MAP kinase inhibitor having a structure (or apharmaceutically acceptable salt thereof) as set forth in the inventionof WO 2004/029040. Hence, a p38 MAP kinase inhibitor having a structure(or a pharmaceutically acceptable salt thereof) as set forth below maybe used:

wherein R₁ and R₂ are independently selected from optionally substitutedaryl and optionally substituted heteroaryl;

-   R₃ is selected from hydrogen, optionally substituted alkyl, —N═CR′″,    —C(O)R′, —C(O)NR′R″, —NR′R″, optionally substituted aryl, optionally    substituted heteroaryl, and optionally substituted heterocycle,    wherein R′ and R″ are independently selected from hydrogen,    optionally substituted alkyl, optionally substituted aryl, and    optionally substituted heterocycle;-   R₄ is selected from hydrogen, optionally substituted alkyl,    optionally substituted aryl, optionally substituted heteroaryl,    optionally substituted heterocycle, and —SiR′″R″″R′″″ wherein R′″,    R″″, and R′″″ are each an independent straight chain or branched    C₁₋₅alkyl, or R3, R4 and the —C—N— to which R3 and R4 are connected    together form an optionally substituted 5- or 6-membered ring;-   R₅ is selected from optionally substituted alkyl, —C(O)OR′,    —C(O)NR′R″, C(O)NHNHC(O)R6, —SO₂NR′R″, —C(O)R′, —NR′R″, nitrile,    nitro, halo, and optionally substituted heterocycle, or R4, R5 and    the —C—N— to which R4 and R5 are connected together form an    optionally substituted 5- or 6-membered ring; and-   R₆ is selected from H, alkyl, optionally substituted aryl; with the    provisos that-   (1) R₁ and R₂ are not both optionally substituted phenyl;-   (2) if either R₁ or R₂ is optionally substituted phenyl or    3-thienyl, and the other is unsubstituted

-    then R3 is not hydrogen, unsubstituted alkyl, —(CH₂)₃OH, —(CH₂)₃PH,    —(CH₂)₃OMs, or —(CH₂)₂N(CH₂)₂O—(CH₂)₂, and R5 is not unsubstituted    alkyl, —(CH₂)₃OH, (CH₂)₃PH, —(CH₂)₃OMS, or —(CH₂)₂N(CH₂)₂O(CH₂)₂;    and-   (3) R₄ does not form a fused ring with both R₃ and R₅.

Preferably, R₁ is substituted with one or more groups selected fromhydrogen, amino, alkyl substituted amino, aryl substituted amino,hydroxy, methoxy, phenyl ether, S-alkyl, halogen, trifluoromethyl, andnitro.

Preferably, R₂ is substituted with one or more groups selected fromhydrogen, amino, alkyl substituted amino, aryl substituted amino,hydroxy, methoxy, phenyl ether, S-alkyl, halogen, trifluoromethyl, andnitro. More preferably, R₂ is heteroaryl having 1-3 N.

Preferably, R₃ is selected from hydrogen, alkyl, aryl, heteroaryl,heterocycle, and —NR′R″, wherein R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and heterocycle.

Preferably, R₄ is hydrogen or alkyl, More preferably, R₄ is hydrogen or30 methyl.

Preferably, R₅ is selected from alkyl, —C(O)OR′, —O(O)NR′R″, nitrile,and heterocycle. In particular, the preferred alkyl is selected from—(CH₂)nOR′, —(CH₂)nNR′R′″, —(CH₂)nCOOR′, and —(CH₂)nCONR′R″; thepreferred NR′R″ group is —NHCOR′; the preferred heterocycles are esterisosteres (e.g. oxadiazole and the like, such as derivatives of1,2,4-triazole, 1,2,4-triazol-3-ol, isoxazol-3-ol,imidazolidine-2,4-dione, 4H-[1,2,4]oxadiazol-5-one,4H-[1,2,4]thiadiazol-5-one, 4H-[1,2,4]oxadiazole-5-thione, oxazole,[1,3,4]oxadiazole).

As used herein, the following chemical terms shall have the meanings asset forth in the following paragraphs: “independently”, when inreference to chemical substituents, shall mean that when more than onesubstituent exists, the substituents may be the same or different.

“Alkyl” shall mean straight, cyclic and branched-chain alkyl. Unlessotherwise stated, the alkyl group will contain 1-20 carbon atoms. Unlessotherwise stated, the alkyl group may be optionally substituted with oneor more groups such as halogen, OH, ON, mercapto, nitro, amino,C1-C5-alkyl, C1-C8-alkoxyl, C1-C8-alkylthio, C1-C8-alkyl-amino,di(C1-C8-alkyl)amino, (mono-, di-, tri-, and per-) halo-alkyl, formyl,carboxy, alkoxycarbonyl, C1-C8-alkyl-CO—O—, C1-C8-alkyl-CO—NH—,carboxamide, hydroxamic acid, sulfonamide, sulfonyl, thiol, aryl,aryl(C1-C8)-alkyl, heterocyclyl, and heteroaryl.

“Alkoxy” shall mean -0-alkyl and unless otherwise stated, it will have1-8 carbon atoms.

“Halogen” or “halo” shall mean fluorine, chlorine, bromine or iodine; 30“PH” or “Ph” shall mean phenyl; “Ac” shall mean acyl; “Bn” shall meanbenzyl; “Me” shall mean methyl.

The term “acyl” as used herein, whether used alone or as part of asubstituent group, means an organic radical having 2 to 6 carbon atoms(branched or straight chain) derived from an organic acid by removal ofthe hydroxyl group. The term “Ac” as used herein, whether used alone oras part of a substituent group, means acetyl.

“Aryl” or “Ar,” whether used alone or as part of a substituent group, isa carbocyclic aromatic radical including, but not limited to, phenyl, I-or 2-naphthyl and the like. The carbocyclic aromatic radical may besubstituted by independent replacement of Ito 5 of the hydrogen atomsthereon with halogen, OH, ON, mercapto, nitro, amino, C1-C8-alkyl,C1-C8-alkoxyl, 01-C8-alkylthio, 01-08-alkyl-amino, di(C1-C8-alkyl)amino,(mono-, di-, tri-, and per-) halo-alkyl, formyl, carboxy,alkoxycarbonyl, C1-C8-alkyl-CO—O—, C1-C8-alkyl-CO—NH—, or carboxamide.Illustrative aryl radicals include, for example, phenyl, naphthyl,biphenyl, fluorophenyl, difluorophenyl, benzyl, benzoyloxyphenyl,carboethoxyphenyl, acetylphenyl, ethoxyphenyl, phenoxyphenyl,hydroxyphenyl, carboxyphenyl, trifluoromethylphenyl, methoxyethylphenyl,acetamidophenyl, tolyl, xylyl, dimethylcarbamyiphenyl and the like. “Ph”or “PH” denotes phenyl.

Whether used alone or as part of a substituent group, “heteroaryl”refers to a cyclic, fully unsaturated radical having from five to tenring atoms of which one ring atom is selected from S, O, and N; 0-2 ringatoms are additional heteroatoms independently selected from S, O, andN; and the remaining ring atoms are carbon. The radical may be joined tothe rest of the molecule via any of the ring atoms. Exemplary heteroarylgroups include, for example, pyridinyl, pyrazinyl, pyrimidinyl,pyridazinyl, pyrroyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isoxazolyl, thiadiazolyl, triazolyl, triazinyl, oxadiazolyl, thienyl,furanyl, quinolinyl, isoquinolinyl, indolyl, isothiazolyl, 2-oxazepinyl,azepinyl, N-30 oxo-pyridyl, I-dioxothienyl, benzothiazolyl,benzoxazolyl, benzothienyl, quinolinyl-N-oxide, benzimidazolyl,benzopyranyl, benzisothiazolyl, benzisoxazolyl, benzodiazinyl,benzofurazanyl, benzothiopyranyl, indazolyl, indolizinyl, benzofuryl,chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl,pyrrolopyridinyl, furopyridinyl (such as fu roII2,3-cjpyridinyl,furo[3,2-bipyridinyl, or furoll-2,3-b]pyridinyl), imidazopyridinyl (suchas imidazo[4,5-b]pyridinyl or imidazo[4,5-c]pyridinyl), naphthyridinyl,phthalazinyl, purinyl, pyridopyridyl, quinazolinyl, thienofuryl,thienopyridyl, thienothienyl, and furyl. The heteroaryl group may besubstituted by independent replacement of 1 to 5 of the hydrogen atomsthereon with halogen, OH, CN, mercapto, nitro, amino, C1-08-alkyl,C1-08-alkoxyl, C1-C8-alkylthio, C1-C8-alkyl-amino, di(C1-C8-alkyl)amino,(mono-, di-, tri-, and per-)halo-alkyl, formyl, carboxy, alkoxycarbonyl,C1-C8-alkyl-CO—O—, C1-C8-alkyl-CO—NH—, or carboxamide. Heteroaryl may besubstituted with a mono-oxo to give for example a 4-oxo-1H-quinoline.

The terms “heterocycle,” “heterocyclic,” and “heterocyclo” refer to anoptionally substituted, fully or partially saturated cyclic group whichis, for example, a 4- to 7-membered monocyclic, 7- to 11-memberedbicyclic, or 10- to 15-membered tricyclic ring system, which has atleast one heteroatom in at least one carbon atom containing ring. Eachring of the heterocyclic group containing a heteroatom may have 1, 2, or3 heteroatoms selected from nitrogen atoms, oxygen atoms, and sulfuratoms, where the nitrogen and sulfur heteroatoms may also optionally beoxidized. The nitrogen atoms may optionally be quaternized. Theheterocyclic group may be attached at any heteroatom or carbon atom.Exemplary monocyclic heterocyclic groups include pyrrolidinyl; oxetanyl;pyrazolinyl; imidazolinyl; imidazolidinyl; oxazolyl; oxazolidinyl;isoxazolinyl; thiazolidinyl; isothiazolidinyl; tetrahydrofuryl;piperidinyl; piperazinyl; 2-oxopiperazinyl; 2-oxopiperidinyl;2-oxopyrrolidinyl; 4-piperidonyl; tetrahydropyranyl;tetrahydrothiopyranyl; tetrahydrothiopyranyl sulfone; morpholinyl;thiomorpholinyl; thiomorpholinyl sulfoxide; thiomorpholinyl sulfone;1,3-dioxolane; dioxanyl; thietanyl; thiiranyl; and the like. Exemplarybicyclic heterocyclic groups include quinuclidinyl;tetrahydroisoquinolinyl; dihydroisoindolyl; dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl); dihydrobenzofuryl; dihydrobenzothienyl;dihydrobenzothiopyranyl; dihydrobenzothiopyranyl sulfone;dihydrobenzopyranyl; indolinyl; isochromanyl; isoindolinyl; piperonyl;tetrahydroquinolinyl; and the like. Preferably, heterocycles areselected from the following groups:

Substituted aryl, substituted heteroaryl, and substituted heterocyclemay also be substituted with a second substituted-aryl, a secondsubstituted-heteroaryl, or a second substituted-heterocycle to give, forexample, 4-pyrazol-1-yl-phenyl or 4-pyridin-2-yl-phenyl. Designatednumbers of carbon atoms (e.g., C1-8) shall refer independently to thenumber of carbon atoms in an alkyl or cycloalkyl moiety or to the alkylportion of a larger substituent in which alkyl appears as its prefixroot.

Unless specified otherwise, it is intended that the definition of anysubstituent or variable at a particular location in a molecule beindependent of its definitions elsewhere in that molecule. It isunderstood that substituents and substitution patterns on the compoundsof this invention can be selected by one of ordinary skill in the art toprovide compounds that are chemically stable and that can be readilysynthesized by techniques known in the art as well as those methods setforth herein.

Where the compounds according to this invention have at least onestereogenic center, they may accordingly exist as enantiomers. Where thecompounds possess two or more stereogenic centers, they may additionallyexist as diastereomers. Furthermore, some of the crystalline forms forthe compounds may exist as polymorphs and as such are intended to beincluded in the present invention. In addition, some of the compoundsmay form solvates with water (i.e., hydrates) or common organicsolvents, and such solvates are also intended to be encompassed withinthe scope of this invention.

Some of the compounds of the present invention may have trans and cisisomers. In addition, where the processes for the preparation of thecompounds according to the invention give rise to mixture ofstereoisomers, these isomers may be separated by conventional techniquessuch as preparative chromatography. The compounds may be prepared as asingle stereoisomer or in racemic form as a mixture of some possiblestereoisomers. The non-racemic forms may be obtained by either synthesisor resolution. The compounds may, for example, be resolved into theircomponents enantiomers by standard techniques, such as the formation ofdiastereomeric pairs by salt formation. The compounds may also beresolved by covalent linkage to a chiral auxiliary, followed bychromatographic separation and/or crystallographic separation, andremoval of the chiral auxiliary. Alternatively, the compounds may beresolved using chiral chromatography.

The p38 MAP kinase inhibitors used in the present invention can beisolated and used as free bases. They can also be isolated and used aspharmaceutically acceptable salts. Examples of such salts includehydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic,fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic,hydroethanesulfonic, benzenesulfonic, oxalic, palmoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic andsaccharic.

An “inhibitor” of a p38 MAP kinase includes any molecule, whichdecreases the activity of the kinase or decreases the protein level ofthe kinase. Thus, a kinase inhibitor can be a molecule, which decreasesactivity of the kinase, e.g., by interfering with interaction of thekinase with another molecule, e.g., its substrate. It can also be amolecule, which decreases expression of the gene encoding the kinase. Aninhibitor can also be an antisense nucleic acid, a ribozyme, anantibody, a dominant negative mutant of the kinase, or a phosphatase.Anti-MAP kinase antibodies are commercially available but can also beprepared according to methods known in the art.

The inhibitor can affect a single p38 MAP kinase isoform, more than oneisoform, or all isoforms of p38 MAP kinase. In a preferred embodiment,the p38 MAP kinase inhibitor preferentially inhibits the alpha and/orthe beta forms of the above-mentioned p38 MAP kinase isoforms.

A “direct inhibitor” of a p38 MAP kinase is an inhibitor, whichinteracts with the kinase or binding partner thereof or with a nucleicacid encoding the kinase.

An “indirect inhibitor” of a p38 MAP kinase is an inhibitor whichinteracts upstream or downstream of the kinase in the regulatory pathwayand which does not interacts with the kinase or binding partner thereofor with a nucleic acid encoding the kinase.

Preferably, the p38 MAP kinase inhibitor is not a tyrosine phosphataseinhibitor.

If acting on p38 MAP kinase directly, in one embodiment the inhibitorshould exhibit an IC₅₀ value of about 5 μM or less, preferably 500 nM orless, more preferably 100 nM or less.

Those skilled in the art can determine whether or not a compound isuseful in the present invention by evaluating its p38 MAP kinaseactivity as well as its relative IC₅₀ value. This evaluation can, forexample, be accomplished through conventional in vitro assays, such asby using one or more of the assays described below. In vitro assaysinclude assays that assess inhibition of kinase or ATPase activity ofactivated p38 MAP kinase. In vitro assays can also assess the ability ofthe inhibitor to bind p38 MAP kinase or to reduce or block an identifieddownstream effect of activated p38 MAP kinase, e.g., cytokine secretion.IC₅₀ values are calculated using the concentration of inhibitor thatcauses a 50% decrease as compared to a control.

A binding assay is a fairly inexpensive and simple in vitro assay torun. Binding of a molecule to p38 MAP kinase, in and of itself, can beinhibitory, due to steric, allosteric or charge-charge interactions. Abinding assay can be performed in solution or on a solid phase using p38MAP kinase or a fragment thereof as a target. By using this as aninitial screen, one can evaluate libraries of compounds for potentialp38 MAP kinase regulatory activity.

The target in a binding assay can be either free in solution, fixed to asupport, or expressed in or on the surface of a cell. A label (e.g.radioactive, fluorescent, quenching, etc.) can be placed on the target,compound, or both to determine the presence or absence of binding. Thisapproach can also be used to conduct a competitive binding assay toassess the inhibition of binding of a target to a natural or artificialsubstrate or binding partner. In any case, one can measure, eitherdirectly or indirectly, the amount of free label versus bound label todetermine binding. There are many known variations and adaptations ofthis approach to minimize interference with binding activity andoptimize signal.

For purposes of in vitro cellular assays, the compounds that representpotential inhibitors of p38 MAP kinase function can be administered to acell in any number of ways.

Preferably, the compound or composition can be added to the medium inwhich the cell is growing, such as tissue culture medium for cells grownin culture. The compound is provided in standard serial dilutions or inan amount determined by analogy to known modulators. Alternatively, thepotential inhibitor can be encoded by a nucleic acid that is introducedinto the cell wherein the cell produces the potential inhibitor itself.

Alternative assays involving in vitro analysis of potential inhibitorsinclude those where cells (e.g., HeLa) transfected with DNA coding forrelevant kinases can be activated with substances such as sorbitol,IL-1, TNF, or PMA. After immunoprecipitation of cell lysates, equalaliquots of immune complexes of the kinases are pre-incubated for anadequate time with a specific concentration of the potential inhibitorfollowed by addition of kinase substrate buffer mix containing labeledATP and GST-ATF2 or MBP. After incubation, kinase reactions areterminated by the addition of SDS loading buffer. Phosphorylatedsubstrate is resolved through SDS-PAGE and visualized and quantitated ina phosphorimager.

The p38 MAP kinase regulation, in terms of phosphorylation and IC₅₀values, can be determined by quantitation. See e.g., Kumar, S. et al.,Biochem. Biophys. Res. Commun. 235: 533-538 (1997).

Other in vitro assays can also assess the production of TNF-α as acorrelation to p38 MAP kinase activity. One such example is a HumanWhole Blood Assay. In this assay, venous blood is collected from, e.g.healthy male volunteers into a heparinized syringe and is used within 2hours of collection. Test compounds are dissolved in 100% DMSO and 1 Itlaliquots of drug concentrations ranging from 0 to 1 mM are dispensedinto quadruplicate wells of a 24-well microtiter plate (Nunclon DeltaSI, Applied Scientific Co., San Francisco, Calif.). Whole blood is addedat a volume of 1 ml/well and the mixture is incubated for 15 minuteswith constant shaking (Titer Plate Shaker, Lab-Line Instruments, Inc.,Melrose Park, Ill.) at a humidified atmosphere of 5% CO₂ at 37 C. Wholeblood is cultured either undiluted or at a final dilution of 1:10 withRPMI 1640 (Gibco 31800+NaHCO₃, Life Technologies, Rockville, Md. andScios, Inc., Sunnyvale, Calif.). At the end of the incubation period, 10ttl of LPS (E. coli 0111:B4, Sigma Chemical Co., St. Louis, Mo.) isadded to each well to a final concentration of 1 or 0.1, μg/ml forundiluted or 1:10 diluted whole blood, respectively. The incubation iscontinued for an additional 2 hours. The reaction is stopped by placingthe microtiter plates in an ice bath, and plasma or cell-free supernatesare collected by centrifugation at 3000 rpm for 10 minutes at 4 C. Theplasma samples are stored at −80 C until assayed for TNF-o levels byELISA, following the directions supplied by Quantikine Human TNF-o assaykit (R & D Systems, Minneapolis, Minn.). IC₅₀ values are calculatedusing the concentration of inhibitor that causes a 50% decrease ascompared to a control.

A similar assay is an Enriched Mononuclear Cell Assay. The enrichedmononuclear cell assay begins with cryopreserved Human Peripheral BloodMononuclear Cells (HPBMCs) (Clonetics Corp.) that are rinsed andresuspended in a warm mixture of cell growth media.

The resuspended cells are then counted and seeded at 1×10⁶ cells/well ina 24-well microtitre plate. The plates are then placed in an incubatorfor an hour to allow the cells to settle in each well. After the cellshave settled, the media is aspirated and new media containing 100 ng/mlof the cytokine stimulatory factor Lipopolysaccharide (LPS) and a testchemical compound is added to each well of the microtiter plate. Thus,each well contains HPBMCs, LPS and a test chemical compound. The cellsare then incubated for 2 hours, and the amount of the cytokine TumorNecrosis Factor Alpha (TNF-α) is measured using an Enzyme LinkedImmunoassay (ELISA). One such ELISA for detecting the levels of TNF-α iscommercially available from R & D Systems. The amount of TNF-αproduction by the HPBMCs in each well is then compared to a control wellto determine whether the chemical compound acts as an inhibitor ofcytokine production.

Formulations

By way of an example, the p38 MAP kinase inhibitor has been formulatedin a simple delivery vehicle. However, the p38 MAP kinase inhibitor orcompound that enhances wound healing by inhibiting the p38 MAP kinasepathway may be lyophilized or incorporated in a gel, cream, biomaterial,sustained release delivery vehicle.

The p38 MAP kinase inhibitor will generally be combined with apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to any pharmaceutical carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition, and which can be administered without unduetoxicity. Suitable carriers can be large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers.Such carriers are well known to those of ordinary skill in the art.Pharmaceutically acceptable carriers in therapeutic compositions caninclude liquids such as water, saline, glycerol and ethanol. Auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, can also be present in such vehicles.Pharmaceutically acceptable salts can also be present in thepharmaceutical composition, e.g. mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like.

Preferably, the at least one p38 MAP kinase inhibitor is provided in theform of a wound dressing. That is to say, at least one p38 MAP kinaseinhibitor is provided in the form of a liquid, semi-solid or solidcomposition for application directly to the surface of a wound, or thecomposition is applied to the surface of, or incorporated into, a solidwound contacting layer such as a wound dressing gauze or film. The wounddressing composition may be provided in the form of a fluid or a gel.The at least one p38 MAP kinase inhibitor may be provided in combinationwith conventional pharmaceutical excipients for topical application to awound. Suitable carriers include: Hydrogels containing cellulosederivatives, including hydroxyethyl cellulose, hydroxymethyl cellulose,carboxymethyl cellulose, hydroxypropylmethyl cellulose and mixturesthereof, and hydrogels containing polyacrylic acid (Carbopols). Suitablecarriers also include creams/ointments used for topical pharmaceuticalpreparations, e.g. creams based on cetomacrogol emulsifying ointment.The above carriers may include alginate (as a thickener or stimulant),preservatives such as benzyl alcohol, buffers to control pH such asdisodium hydrogen phosphate/sodium dihydrogen phosphate, agents toadjust osmolarity such as sodium chloride, and stabilisers such as EDTA.

In one embodiment of the invention, the wound dressing composition maybe a slow release solid composition, in which the at least one p38 MAPkinase inhibitor is dispersed in a slow release solid matrix such as amatrix of alginate, collagen, or a synthetic bioabsorbable polymer.Preferably, the wound dressing composition is sterile. The term “wounddressing” in this specification refers to a dressing for topicalapplication to a wound and excludes compositions suitable for systemicadministration. For example, the at least one p38 MAP kinase inhibitormay be dispersed in or on a solid sheet of wound contacting materialsuch as a woven or nonwoven textile material, or may be dispersed in alayer of foam such as polyurethane foam, or in a hydrogel such as apolyurethane hydrogel, a polyacrylate hydrogel, gelatin, carboxymethylcellulose, pectin, alginate, and/or hyaluronic acid hydrogel, forexample in a gel or ointment. In preferred embodiments the at least onep38 MAP kinase inhibitor is dispersed in or on a biodegradable sheetmaterial that provides sustained release of the active ingredient intothe wound, for example a sheet of freeze-dried collagen, freeze-driedcollagen/alginate mixtures (available under the Registered Trade MarkFIBRACOL from Johnson & Johnson Medical Limited) or freeze-driedcollagen/oxidized regenerated cellulose (available under the RegisteredTrade Mark PROMOGRAN from Johnson & Johnson Medical Limited).

As an alternative to topical administration, the p38 MAP kinaseinhibitor may be administered systemically.

Suitably, the patient is also administered an additional wound healingagent (see below).

Dose

The effective dose for a given situation can be determined by routineexperimentation and is within the judgement of the skilled person. Forexample, in order to formulate a range of dosage values, cell cultureassays and animal studies can be used. The dosage of such compoundspreferably lies within the dose that is therapeutically effective in 50%of the population, and that exhibits little or no toxicity at thislevel.

Additional Wound Healing Agents

Optionally, one or more other, conventional wound healing agents (e.g.growth factors, peptides, proteolytic inhibitors, extracellular matrixcomponents, fragments and peptides, steroids, cytokines, oxygen donatorsor vitamins) may also be used in the manufacture of the medicament andcompositions according to the invention. Such conventional wound healingagents may also be used in the method of the present invention. Theinclusion of these agents may allow a synergistic effect on woundhealing. Such additional wound healing agent(s) may be administeredseparately, simultaneously or sequentially with the p38 MAP kinaseinhibitor.

Thus, in one embodiment an effective dose of the p38 MAP kinaseinhibitor or a compound that enhances wound healing by inhibiting thep38 MAP kinase pathway may delivered in conjunction with or alternatingwith another effective wound healing agent from the following groupse.g. growth factors, peptides, proteolytic inhibitors, extracellularmatrix components, fragments and peptides, steroids, cytokines, oxygendonators and vitamins.

In one embodiment the patient may be administered the p38 MAP kinaseinhibitor and the additional wound healing agent(s) by means of a singlemedicament which comprises both the p38 MAP kinase inhibitor and theadditional wound healing agent(s).

In another embodiment the patient is administered the p38 MAP kinaseinhibitor and the additional wound healing agent(s) separately.

In one embodiment of the invention a kit is provided which comprises apharmaceutical composition according to the third aspect of theinvention and a further composition comprising one more additional woundhealing agents.

Type of Wounds

The wounds that the p38 MAP kinase inhibitor or a compound that enhanceswound healing by inhibiting the p38 MAP kinase pathway, may be anexternal wound e.g. diabetic foot ulcer, venous leg ulcer, pressuresore, compromised surgical wound, surgical wound, acute wound, or aninternal wound e.g. adhesions, liver cirrhosis.

The four types of wound healing are primary, delayed primary, partialthickness, and secondary. Secondary healing, also called healing bycontraction, reduces the area of a healing wound. Contraction requiresthe formation of granulation tissue, which is rich in the blood vesselsand fibroblasts which are needed to fill the wound defect. The processinvolves complex interactions between specialized contractingfibroblasts (myofibroblasts) and the wound matrix. It does not usuallyrequired epitheliation. The results are usually cosmetically andfunctionally acceptable. However, if the process is very excessive itcan result in the pathologic process known as wound contracture. Thisprocess is characterized by severe deformities and loss of function.

In one embodiment, the wound is not a wound which is subject to woundcontracture. Examples of wounds, which are not subject to woundcontractures, include small area wounds, partial thickness wounds, acutesurgical incisions and chronic wounds including diabetic foot ulcers,pressure sores, venous leg ulcers and non-healing surgical wounds.Preferably, the wound is not a burn or a surgical wound.

Modes of Delivery

An effective dose of the p38 MAP kinase inhibitor or a compound thatenhances wound healing by inhibiting the p38 MAP kinase pathway may beapplied intravenously, intradermally, subcutaneously, topically(peripherally or directly) to a wound. In one embodiment a wash solutioncomprising the p38 MAP kinase inhibitor can be used locally to promotehealing of the wound.

The Patient

The patient will usually be a mammal, such as a horse (e.g. aracehorse), a dog (e.g. greyhound), a cow, etc. Preferred patients arehumans, including children, adults and the elderly.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

EXAMPLES Example 1

Female diabetic mice (C57BLKs/Bom db/db; B&M, Denmark) agedapproximately 16 weeks will be used in this study. On arrival mice werehoused in groups of 5 to 10 according to Home Office regulations. On thefirst day of the study period, animals were housed in individual cages(cage dimensions 35×15×15 cm with sawdust bedding, changed twiceweekly), in an environment maintained at an ambient temperature of 23°C. with 12-hour light/dark cycles. They were provided with food(Standard Rodent Diet) and water ad libitum. To acclimatise the animalsto their surroundings, prior to experimentation, they were housed for aminimum of one week without disturbance, other than to refresh theirbedding and to replenish their food and water provisions. Followingwounding, animals were monitored until they had fully recovered from theprocedure and then housed under individual conditions for the remainderof the study period. All animal procedures were carried out in a HomeOffice licensed establishment in accordance with Home Office Licenseprocedures.

Animals were anaesthetised (halothane and air) and shaved. A singlestandardised full thickness wound (7.5 mm×7.5 mm) was created in theflank skin of each experimental animal. Wounds received, saline [50 μl],vehicle [50 μl pH 4.0 vehicle], 1 μg of p38 MAP kinase inhibitor [in 50μl pH 4.0 vehicle] or 10 μg of p38 MAP kinase inhibitor [in 50 μl pH 4.0vehicle]. The p38 MAP kinase inhibitor had the following formula:

All wounds were secondarily dressed with a 1.5×1.5 cm pad of RELEASEdressing (Johnson & Johnson Wound Management). The RELEASE pad was heldin place using a circumferential band of the occlusive film dressingBioclusive (Johnson & Johnson Wound Management). Animals werere-anaesthetized and treatments re-applied on days 2, 4, 7, 9 and11-post wounding. Immediately after wounding and subsequently on days 2,4, 7, 9, 11 and 14 all wounds were digitally photographed together witha calibration/identity plate. On day 14 of the study animals will beeuthanised.

Image Pro image analysis software (version 4.1.0.0, Media Cybernetics,USA) was used to calculate wound closure from wound images in each ofthe experimental groups over time.

TABLE 1 Effect of p38 MAP kinase inhibitor or control treatments on thepercentage wound area remaining at days, 2, 4, 7, 9, and 11, relative today 0. Data are presented as the mean of six wounds per treatment ± thestandard error of the mean. % wound area remaining relative to day 0(mean ± standard error) Days post-wounding Treatment 2 4 7 9 11 10.0 μg75.48 ± 6.32 58.34 ± 6.49 29.80 ± 4.66 13.31 ± 1.64 3.34 ± 0.96  1.0 μg86.15 ± 6.63 66.32 ± 6.58 38.65 ± 2.62 18.87 ± 4.44 6.03 ± 2.17 citratebuffer 90.88 ± 4.15 74.50 ± 3.95 41.59 ± 5.27 23.91 ± 4.58 7.07 ± 1.52Saline 97.43 ± 7.00 80.64 ± 7.98 48.18 ± 6.09 24.56 ± 4.42 7.19 ± 1.61

Non-parametric analysis (Kruskal Wallis—Multivariate Analysis followedby ad hoc two sample Mann Whitney U-test analysis) will be used to testthe significance of any inter-group differences in wound closure.

When wound closure consequent to treatment with the delivery vehicle(Citrate Buffer, pH 4.0) was compared to that consequent to salinetreatment—no statistically significant differences were noted.

Treatment with p38 MAP kinase inhibitor appeared to accelerate theclosure of diabetic wounds when compared to treatment with the vehicle(Citrate Buffer, pH 4.0). This effect was most apparent for the highestdose of this agent, with wounds treated with 10.0 μg p38 MAP kinaseinhibitor found to be statistically smaller than citrate treated woundsat the day 9 and day 11 assessment points.

When closure in response to treatment with 10.0 μg of p38 MAP kinaseinhibitor was compared to that in response to saline treatment, closurewas found to be noticeably more rapid in the 10.0 μg group at themajority of the assessment points (day 2, p=0.055; day 7, p=0.055; day11, p=0.055); an effect that achieved statistical significance at the 9day assessment point (p=0.025).

Example 2

Human Adult Dermal Fibroblasts isolated from a 34 year old male donor(CRL 2068; American Type Culture Collection) were grown to 95%confluency in 75 cm² vented, tissue culture flasks (Corning). Cells wereroutinely passaged in 0.05% trypsin/EDTA and resuspended in Dubelco'sModified Eagle's Medium (DMEM) (Gibco) supplemented with 10% bovinefetal calf serum and penicillin/streptomycin (Gibco).

Cells were re-suspended at a density of 140,000/ml (4× the densityrequired for the final collagen gel). DMEM, containing 10% FCS, wasmixed with cells [140,000 cells/ml] in DMEM, containing 10% FCS, and Rattail collagen Type I [final conc=1 mg/ml] at a ratio of 2:1:1. Thissolution was then aliquoted into 12 well, tissue culture plates(Corning) at 1 ml/well and allowed to solidify in an incubator at 37° C.and 5% CO₂ for 1 hour. Once the gel has polymerised, the gels arecarefully detached from the rim of each gel and the vehicle or p38 MAPkinase inhibitor treatments added at time 0. The p38 MAP kinaseinhibitor used in this example was the same as in Example 1. FPCLs werechallenged with either vehicle [1.0 ml/well of DMEM, containing 10% FCSv/v, antibiotics and vehicle (10% v/v)] or p38 MAP kinase inhibitor [1.0ml/well of DMEM, containing 10% FCS v/v, antibiotics and vehicle (10%v/v) containing 20 μg p38 MAP kinase inhibitor].

The fibroblast-populate collagen lattices (FPCL) were then photographedusing video image capture [JVC 3 CCD fitted with a 18-108/25 Zoom Lens]and analysed by Image Pro 5.0 at time 0, 1, 2 and 3 days. Data arepresented as the mean of 3 wells per treatment ± the standard error ofthe mean.

1. A method of promoting wound healing of an external wound in a patientcomprising administering a therapeutically effective amount of a p38 MAPkinase inhibitor or a pharmaceutically acceptable salt thereof, whereinthe inhibitor has the following structure:

wherein said external wound is not a burn.
 2. The method according toclaim 1 wherein an additional wound healing agent is administered to thepatient.
 3. The method according to claim 2 wherein the additional woundhealing agent is administered to the patient in a composition comprisingthe p38 MAP kinase inhibitor and the additional wound healing agent. 4.The method according to claim 2 wherein the additional wound healingagent is administered to the patient in a composition which is separatefrom the p38 MAP kinase inhibitor.
 5. The method according to claim 1wherein the p38 MAP kinase inhibitor is provided in the form of a wounddressing.
 6. The method of claim 1 wherein the p38 MAP kinase inhibitoris administered systemically.
 7. The method of claim 1, wherein thepharmaceutically acceptable salt is at least one member selected fromthe group consisting of hydrobromic, hydroiodic, hydrochloric,perchloric, sulfuric, maleic, fumaric, malic, tartaric, citric, benzoic,mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic,palmoic, 2 -naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,saccharic, and mixtures thereof.